γ射线诱导质粒pGEM-3Zf(-)DNA lacZ基因突变的序列分析

利用质粒ｐＧＥＭ－３Ｚｆ（－）ＤＮＡ上带有的ｌａｃＺ基因,通过Ｘ－ｇａｌ显色平板筛选经＾６０Ｃｏγ辐射诱导形成的突变体。对４个突变体的ｌａｃＺ基因进行序列分析发现,所有突变体无一例外地检测到碱基变化。在被测序的４８９ｂｐ范围内,一个突变体发生的碱基变异达２－７个位点。碱基变异的类型包括颠换（５７％）、转换（１９％）、缺失（１９％）及插入（５％）。在碱基置换中,以Ｃ／Ｇ→Ａ／Ｔ颠换最多（占５０％）。     

AT—AC内含子及其剪接机理的研究进展

ＡＴ┐ＡＣ内含子及其剪接机理的研究进展滕胜明镇寰（杭州大学生命科学学院,杭州３１００１２）关键词ＡＴ－ＡＣ内含子剪接机理最近发现了一种新类型的内含子,它在ｍＲＮＡ前体中存在的比例不到０．１％,其剪接位点高度保守的双核苷酸为ＡＴ和ＡＣ（ＡＴ－ＡＣ内含子...     

缺氧时培养的心内膜内皮细胞内皮素自分泌调节的探讨

本实验观察缺氧对心内膜内皮细胞（ＥＥＣ）内皮素－１（ＥＴ－１）分泌的影响。传代培养的新生小牛右心室ＥＥＣ的ＥＴ－１免疫组织化学显色强阳性。采用放免测定发现ＥＥＣ可向培养液中分泌ＥＴ－１,其分泌速度与细胞密度呈线性负相关（ｒ＝－０．９５４２,Ｐ＜０．００１）,与温育时间呈指数负相关（ｒ＝－０．９９８,Ｐ＜０．００１）。０％Ｏ２缺氧６～１２ｈ后,ＥＥＣ的ＥＴ－１分泌约增加１倍（Ｐ＜０．００１）。无论在常氧还是缺氧情况下,硝普钠抑制ＥＥＣ的ＥＴ－１分泌,而ＮＯ合酶抑制剂ＬＮＡ则促进ＥＴ－１分泌。上述结果表明：ＥＥＣ可能通过分泌ＥＴ－１调节心脏功能,内源性ＮＯ抑制ＥＴ－１分泌;缺氧可能显著影响ＥＥＣ的ＥＴ－１分泌     

内皮素和bFGF对MC的促增殖作用及胰岛素的协同效应

采用３Ｈ－ＴｄＲ参入法,测定碱性成纤维细胞生长因子（ｂＦＧＦ）、胰岛素和内皮素－１（ＥＴ－１）对体外培养的大鼠肾小球系膜细胞（ＭＣ）增殖的影响,以及胰岛素与ｂＦＧＦ或ＥＴ－１促ＭＣ增殖的协同作用。结果表明,不同浓度的ｂＦＧＦ（５－２００ｎｇ／ｍｌ）和胰岛素（０．１－２．４Ｕ／ｍｌ）均显著升高ＭＣ的３Ｈ－ＴｄＲ参入值（ｃｐｍ值）（Ｐ＜０．０１）。ＥＴ－１对ＭＣ的ｃｐｍ值的影响依剂量不同呈现两种不同的效应,在１０－９－１０－７ｍｏｌ／Ｌ时,随着浓度的升高,ＭＣ的ｃｐｍ值明显升高（Ｐ＜０．０１）,并以１０－８ｍｏｌ／Ｌ作用最强;当升高到１０－６ｍｏｌ／Ｌ时,ＭＣ的ｃｐｍ值出现降低趋势。胰岛素与ｂＦＧＦ或低浓度ＥＴ－１（≤１０－８ｍｏｌ／Ｌ）共同作用于ＭＣ时,ＭＣ的ｃｐｍ值明显高于二者单独作用之和（Ｐ＜０．０１）,与高浓度ＥＴ－１（＞１０－７ｍｏｌ／Ｌ）共同作用于ＭＣ时,ＭＣ的ｃｐｍ值小于二者单独作用之和（Ｐ＞０．０５）。上述结果说明,胰岛素、ｂＦＧＦ和ＥＴ－１均能显著促进ＭＣ增殖;胰岛素与ｂＦＧＦ或低浓度的ＥＴ－１促ＭＣ增殖具有正协同作用,与高浓度ＥＴ－１呈现负协同作用。     

天花粉蛋白Glu160在N-糖苷酶活性中的作用

用浸泡法得到了（Ｅ１６０Ａ）天花粉蛋白（ｔｒｉｃｈｏｓａｎｔｈｉｎ, ＴＣＳ）,（Ｅ１６０Ｄ）ＴＣＳ与Ａｄｅ 和（Ｅ１６０Ａ）ＴＣＳ与ＦＭＰ复合物的晶体．在Ｍａｒ Ｒｅｓｅａｒｃｈ 面探测器系统上分别收集了０．２０ ｎｍ ,０．１９ｎｍ 和０．２０５ ｎｍ 分辨率的Ｘ 射线衍射数据,数据处理用Ｍａｒ Ｓｃａｌｅ 程序系统完成．用同晶差值Ｆｏｕｒｉｅｒ法解析了（Ｅ１６０Ａ）ＴＣＳ－Ａｄｅ,（Ｅ１６０Ｄ）ＴＣＳ－Ａｄｅ 和（Ｅ１６０Ａ）ＴＣＳ－ＦＭＰ的晶体结构,结构修正利用Ｘ－ＰＬＯＲ程序,修正结果,晶体学Ｒ因子分别为０．１６６,０．１７６,０．１７９．键长和键角的ＲＭＳ偏差分别为０．００１０ ｎｍ 和２．５０３°,０．００１３ ｎｍ 和２．６６５°,０．００１２ ｎｍ 和２．６７６°．在这三个结构中均未见到Ｇｌｕ１８９侧链方向的改变．Ａｄｅ 或ＦＭＰ仍结合在Ｎ－糖苷酶活性口袋之中,它夹在Ｔｙｒ７０和Ｔｙｒ１１１两个侧链环之间,与Ｔｙｒ７０环近乎平行．这一结果表明：ＴＣＳ中的Ｇｌｕ１６０分别突变成Ａｌａ 和Ａｓｐ,仍能与ＡＭＰ发生Ｎ－糖苷酶反应,但是活性降低了一些．可见Ｇｌｕ１６０对ＴＣＳ与ＡＭＰ的作用是重要的,但不是必要的．     

黑柄炭角菌的单核苷酸多态性研究

以SNPs作为分子标记,对黑柄炭角菌的生活史和遗传多样性进行研究。基于黑柄炭角菌的基因组文库,在两个菌株（X-WC和X-LY）的34个片段（19,680bp）中共发现193个SNPs,SNP发生率为0.981%。193个SNPs位点都有两个可变的核苷酸,其中132个转换,61个颠换,转换与颠换比率为2.16。此外,对两个菌株序列进行比较,发现了41个插入/缺失位点。从34个片段中选择4个片段,分别比较菌株内的SNPs情况。X-WC的菌株内SNP发生率为每个核苷酸1.308%（30/2293）,X-LY的菌     

基于ITS和matK基因对牡丹组序列分析及其亲缘关系的研究

以芍药属牡丹组全部9个野生种、5个紫斑牡丹栽培品种及3个中原牡丹品种为试材,进行核糖体内转录间隔区（ITS）和叶绿体成熟酶K （matK）基因片段测序分析,探讨ITS和matK序列为牡丹组所有野生种种间关系提供分子证据。从GeneBank中选取了1个牡丹及3个外类群芍药、川赤芍和草芍药的ITS及matK序列。对试验样品进行DNA提取、PCR扩增并双向测序得到44条序列,人工校正后将所得44条序列进行比对;计算碱基组成频率、变异位点、简约信息位点数、转换/颠换比率、种内及种间遗传距离,以邻接法进行系统发育分析。结果表明,牡丹组所有个体ITS序列长度在750~800 bp,含有86个多态位点,74个简约信息位点,转换/颠换比率（R）为1.2;而matK序列含有20个简约信息位点,转换/颠换比率（R）为1.7。ITS序列分析将牡丹组野生种分为两大枝,稷山牡丹、紫斑牡丹、卵叶牡丹和杨山牡丹聚为一枝,狭叶牡丹、滇牡丹、黄牡丹和大花黄牡丹聚为另一枝,这两枝分别与革质花盘亚组和肉质花盘亚组相对应,而四川牡丹位于革质花盘亚组最底端,支持前人研究将四川牡丹归为革质花盘亚组。matK序列分析的牡丹组野生种间遗传距离结果不理想,未能清晰的表明野生种之间的亲缘关系。由此说明,ITS序列更适合牡丹组野生种间亲缘关系的研究分析。     

EDRF对PE引起的大鼠主动脉缩血管效应的作用

本文研究ＥＤＲＦ（ｅｎｄｏｔｈｅｌｉｕｍ－ｄｅｒｉｖｅｄｒｅｌａｘｉｎｇｆａｃｔｏｒ,ＥＤＲＦ）对ＰＥ（ｐｈｅｎｙｌｅｐｈｒｉｎｅ）引起的大鼠主动脉收缩反应的影响。内皮完整和去内皮的大鼠主动脉环悬挂于器官浴槽中,测定血管的张力和收缩速度的变化。所有的实验在消炎痛（ｉｎｄｏｍｅｔｈａｃｉｎ,１０μｍｏｌ／Ｌ）存在下进行。用美兰（ｍｅｔｈｙｌｅｎｅｂｌｕｅ,ＭＢ,１０μｍｏｌ／Ｌ）或左旋硝基精氨酸（ＮＧ－ｎｉｔｒｏ－Ｌ－ａｒｇｉｎｉｎｅ,Ｌ－ＮＮＡ,３０μｍｏｌ／Ｌ）处理内皮完整的大鼠主动脉环,ＰＥ的剂量－收缩张力曲线明显左移,ＥＣ３０值均降低５倍,最大反应比率分别为１．６±０．４和１．６±０．５。在去内皮的大鼠主动脉环中,经ＭＢ和Ｌ－ＮＮＡ处理后,仍可见ＥＣ３０下降３倍,最大反应比率均为１．０±０．２。后者可能与血管平滑肌产生少量ＥＤＲＦ有关。我们的结果提示ＰＥ对血管的收缩反应也受血管内皮和平滑肌产生的ＥＤＲＦ的调控     

普氏野马线粒体DNAD—loop区序列多态性研究

目的：了解中国新疆吉木萨尔野马繁殖中心的普氏野马（Equus przewalskii）遗传多样性及其遗传背景。方法：采用PCR产物直接测序法,对15匹普氏野马线粒体DNAD—loop高变区进行测序分析。结果：测定15个个体的线粒体DNAD—loop高变区15464—15866片段序列402bp。检测到12种单倍型,包括37个多态位点,占全部序列的9．2％,其中转换位点2,4个、颠换位点20个、转换位点和颠换并存位点8个、缺失位点3个。A％＋T％含量（56．1％）高于G％＋C％含量（43．9％）,平均A含量为28．4％,T含量为27．7％,C含量为29％,G含为14．9％。单倍型间平均遗传距离为0．030,单倍型多态性（h）为1±0．00116,核苷酸多态性（7c）为2．90％。15匹普氏野马线粒体DNAD—loop高变区之间平均核苷酸变异率为2．48％。结论：研究表明我国新疆吉木萨尔野马繁殖中心的普氏野马线粒体DNAD—loop区序列存在丰富的多态性。     

卵磷脂胆固醇酰基转移酶缺乏综合征

卵磷脂胆固醇酰基转移酶（ｌｅｃｉｔｈｉｎｃｈｏｌｅｓｔｅｒｏｌａｃｙｌｔｒａｎｓｆｅｒａｓｅ,ＬＣＡＴ）缺乏综合征是一组独特的有关ＨＤＬ代谢的遗传性疾病,由ＬＣＡＴ基因的自发突变所致。近几年来,这方面的研究很多,特别是与动脉粥样硬化之间的关系引起人们的广泛关注。１．ＬＣＡＴ基因与蛋白质人类ＬＣＡＴ基因位于１６ｑ２１－２２区,含有４．２ｋｂ的碱基对,主要在肝脏表达。其成熟蛋白质分子量为４７．０９０ｋＤ,除含有一段极其疏水的多肽外,还含有４个Ｎ－连接的糖基化作用位点,这些位点的消除对酶的活性影响很大…     

Variation in the Pattern of Nucleotide Substitution Across Sites

A model of nucleotide substitution that allows the transition/transversion rate bias to vary across sites was constructed. We examined the fit of this model using likelihood-ratio tests by analyzing 13 protein coding genes and 1 pseudogene. Likelihood-ratio testing indicated that a model that allows variation in the transition/transversion rate bias across sites provided a significant improvement in fit for most protein coding genes but not for the pseudogene. When the analysis was repeated with parameters estimated separately for first, second, and third codon positions, strong heterogeneity was uncovered for the first and second codon positions; the variation in the transition/transversion rate was generally weaker at the third codon position. The transition rate bias and branch lengths are underestimated when variation in the transition/transversion rate was not accommodated, suggesting that it may be important to accommodate variation in the pattern of nucleotide substitution for accurate estimation of evolutionary parameters. Received: 4 November 1997 / Accepted: 19 May 1998     

The Genetic Code Is One in a Million

Statistical and biochemical studies of the genetic code have found evidence of nonrandom patterns in the distribution of codon assignments. It has, for example, been shown that the code minimizes the effects of point mutation or mistranslation: erroneous codons are either synonymous or code for an amino acid with chemical properties very similar to those of the one that would have been present had the error not occurred. This work has suggested that the second base of codons is less efficient in this respect, by about three orders of magnitude, than the first and third bases. These results are based on the assumption that all forms of error at all bases are equally likely. We extend this work to investigate (1) the effect of weighting transition errors differently from transversion errors and (2) the effect of weighting each base differently, depending on reported mistranslation biases. We find that if the bias affects all codon positions equally, as might be expected were the code adapted to a mutational environment with transition/transversion bias, then any reasonable transition/transversion bias increases the relative efficiency of the second base by an order of magnitude. In addition, if we employ weightings to allow for biases in translation, then only 1 in every million random alternative codes generated is more efficient than the natural code. We thus conclude not only that the natural genetic code is extremely efficient at minimizing the effects of errors, but also that its structure reflects biases in these errors, as might be expected were the code the product of selection. Received: 25 July 1997 / Accepted: 9 January 1998     

Estimating synonymous and nonsynonymous substitution rates under realistic evolutionary models

Approximate methods for estimating the numbers of synonymous and nonsynonymous substitutions between two DNA sequences involve three steps: counting of synonymous and nonsynonymous sites in the two sequences, counting of synonymous and nonsynonymous differences between the two sequences, and correcting for multiple substitutions at the same site. We examine complexities involved in those steps and propose a new approximate method that takes into account two major features of DNA sequence evolution: transition/transversion rate bias and base/codon frequency bias. We compare the new method with maximum likelihood, as well as several other approximate methods, by examining infinitely long sequences, performing computer simulations, and analyzing a real data set. The results suggest that when there are transition/transversion rate biases and base/codon frequency biases, previously described approximate methods for estimating the nonsynonymous/synonymous rate ratio may involve serious biases, and the bias can be both positive and negative. The new method is, in general, superior to earlier approximate methods and may be useful for analyzing large data sets, although maximum likelihood appears to always be the method of choice.     

Rates and Patterns of Base Change in the Small Subunit Ribosomal RNA Gene

The small subunit ribosomal RNA gene (srDNA) has been used extensively for phylogenetic analyses. One common assumption in these analyses is that substitution rates are biased toward transitions. We have developed a simple method for estimating relative rates of base change that does not assume rate constancy and takes into account base composition biases in different structures and taxa. We have applied this method to srDNA sequences from taxa with a noncontroversial phylogeny to measure relative rates of evolution in various structural regions of srRNA and relative rates of the different transitions and transversions. We find that: (1) the long single-stranded regions of the RNA molecule evolve slowest, (2) biases in base composition associated with structure and phylogenetic position exist, and (3) the srDNAs studied lack a consistent transition/transversion bias. We have made suggestions based on these findings for refinement of phylogenetic analyses using srDNA data.     

Are transversion mutations better? A Mutagenesis Assistant Program analysis on P450 BM-3 heme domain

Directed evolution represents a versatile tool to tailor enzyme properties to needs in industrial applications and to understand structure-function relationships. Genetic diversity is commonly generated using error-prone PCR. Exploration of sequence space by random mutagenesis strongly favors transitions when enzyme-based mutagenesis methods are employed (Wong, T. S., Zhurina, D., Schwaneberg, U., Comb. Chem. High Throughput Screen. 2006, 9, 271-288). The genetic code has been organized in a manner that limits chemical diversity when a single transition mutation occurs in a codon (Wong, T. S., Roccatano, D., Schwaneberg, U., Biocatal. Biotransformation 2006, in press). Are transitions more beneficial than transversions for adapting biocatalysts to non-natural process conditions? In a statistical analysis performed with the Mutagenesis Assistant Program (MAP), we compared the consequences of transition and transversion bias on amino acid substitution patterns of the P450 BM-3 heme domain. For the analysis, we used a recently introduced benchmarking system consisting of a protein structure indicator, an amino acid diversity indicator with a codon diversity coefficient, and a chemical diversity indicator. A detailed analysis for the P450 BM-3 heme domain showed that an ideal transversion bias generates more diverse amino acid substitution patterns with a significantly different chemical composition than an ideal transition bias. Emphasis is given on the theoretical analysis with a brief discussion on potential implication of transition and transversion bias in directed evolution experiments.     

氟氏链霉菌离子束注入突变谱的分析

用低能N⁺离子束注入转谷氨酰胺酶产生菌氟氏链霉菌后,通过试验,初步确定了注入的效应曲线,获得了一系列突变菌株。提取原始菌株和突变菌株的DNA,采用PCR反应分段扩增出转谷氨酰胺酶基因进行单链构象多态性分析(SSCP),并将特异性条带克隆测序进行基因突变型的鉴定,分析离子束注入引起链霉菌基因的基因突变类型及特点。结果显示:碱基变异的类型包括转换、颠换和缺失。在检测到的24个碱基突变中,主要是碱基的置换(87.5%),碱基缺失的比例比较小(12.5%)。在碱基置换中,转换的频率(58.3%)高于颠换的频率(29.2%)。转换主要以C→T,A→G为主,颠换以G→T,C→G为主。此外构成DNA的4种碱基均可以被离子束辐照诱发变异,其中胞嘧啶发生突变的频率较高。     

嗜盐古生菌br基因的遗传分析

从新疆阿尔金山地区阿乌拉仔盐湖分离纯化到几株极端嗜盐古生菌AJ11, AJ12和AJ13, 采用PCR技术分别扩增了其16S rRNA基因(16S rDNA)和编码螺旋C至螺旋G的细菌视紫红质(bacteriorhodopsin, BR)蛋白基因片段, 测定了基因的核苷酸序列。基于16S rDNA序列的同源性比较以及系统发育学研究表明, 分离到的菌株是Natrinema属中成员, 并构成一个独立的微生物种群。随后的遗传分析, 包括GC含量、转换与颠换的比率、同义突变率分析, 表明br基因间具有较高的遗传分歧程度, 并面临着净化选择和偏倚突变压的双重抑制。研究为物种资源及BR蛋白资源的进一步利用打下基础。     

Transition-transversion bias is not universal: a counter example from grasshopper pseudogenes

Comparisons of the DNA sequences of metazoa show an excess of transitional over transversional substitutions. Part of this bias is due to the relatively high rate of mutation of methylated cytosines to thymine. Postmutation processes also introduce a bias, particularly selection for codon-usage bias in coding regions. It is generally assumed, however, that there is a universal bias in favour of transitions over transversions, possibly as a result of the underlying chemistry of mutation. Surprisingly, this underlying trend has been evaluated only in two types of metazoan, namely Drosophila and the Mammalia. Here, we investigate a third group, and find no such bias. We characterize the point substitution spectrum in Podisma pedestris, a grasshopper species with a very large genome. The accumulation of mutations was surveyed in two pseudogene families, nuclear mitochondrial and ribosomal DNA sequences. The cytosine-guanine (CpG) dinucleotides exhibit the high transition frequencies expected of methylated sites. The transition rate at other cytosine residues is significantly lower. After accounting for this methylation effect, there is no significant difference between transition and transversion rates. These results contrast with reports from other taxa and lead us to reject the hypothesis of a universal transition/transversion bias. Instead we suggest fundamental interspecific differences in point substitution processes.     

Mammalian mitochondrial DNA evolution: A comparison of the cytochrome b and cytochrome c oxidase II genes

The evolution of two mitochondrial genes, cytochrome b and cytochrome c oxidase subunit II, was examined in several eutherian mammal orders, with special emphasis on the orders Artiodactyla and Rodentia. When analyzed using both maximum parsimony, with either equal or unequal character weighting, and neighbor joining, neither gene performed with a high degree of consistency in terms of the phylogenetic hypotheses supported. The phylogenetic inconsistencies observed for both these genes may be the result of several factors including differences in the rate of nucleotide substitution among particular lineages (especially between orders), base composition bias, transition/transversion bias, differences in codon usage, and different constraints and levels of homoplasy associated with first, second, and third codon positions. We discuss the implications of these findings for the molecular systematics of mammals, especially as they relate to recent hypotheses concerning the polyphyly of the order Rodentia, relationships among the Artiodactyla, and various interordinal relationships.Correspondence to: R.L. Honeycutt     

Patterns of transitional mutation biases within and among mammalian genomes

Significant transition/transversion mutation bias is a well-appreciated aspect of mammalian nuclear genomes; however, patterns of bias among genes within a genome and among species remain largely uncharacterized. Understanding these patterns is important for understanding similarities and differences in mutational patterns among genomes and genomic regions. Therefore, we have conducted an analysis of 7,587 pairs of sequences of 4,347 mammalian protein-coding genes from seven species (human, mouse, rat, cow, sheep, pig, and macaque) and from the introns of 51 gene pairs and multiple intergenic regions (37 kbp, 52 kbp and 65 kbp) from the human, chimpanzee, and baboon genomes. Our analyses show that genes and regions with widely varying base composition exhibit uniformity of transition mutation rate both within and among mammalian lineages, as long as the transitional mutations caused by CpG hypermutability are excluded. The estimates show no relationship to potential intrachromosomal or interchromosomal effects. This uniformity points to similarity in point mutation processes in genomic regions with substantially different GC-content biases.